We propose to continue our studies of the molecular basis of the replication and gene expression of the hepatitis B viruses of man and animals. Our efforts will continue to center on the ground squirrel hepatitis virus (GSHV) as a model system in which to pursue the following objectives: (1) to develop a genetic system for mutational analysis of essential viral genes (2) to isolate and characterize the protein products of these genes (3) to elucidate the structures and coding potential of viral mRNA's, and to identify the viral transcript which serves as the putative template for reverse transcription (4) to identify and characterize transcriptional regulatory signals governing the production of these RNA's and (5) to develop cell culture systems for the analysis of viral replication and gene expression. Our program involves the isolation and characterization of intrahepatic DNA and RNA using subcellular fractionation, blot-hybridization, S1 nuclease mapping and molecular cloning techniques. Viral proteins will be identified and studied using antisera raised to synthetic peptides and fusion proteins generated in prokaryotic hosts. The ability of cloned DNA to initiate infection of animal hosts will be used to score the phenotype of mutant or recombinant genomes generated in vitro, as a functional test of the activity of previously uncharacterized viral genes.